Blood substitute has recently received a great deal of attention due to its potential application to solving the blood shortage problems as well as providing an expeditious means allowing blood transfusion during wars or other emergency situations. Blood substitute can also find applications in many other situations, such as in preventing the spreading of diseases such as AIDS, hepatitis B, etc.; in the treatment of certain blood diseases; for organ preservation prior to organ transplants: in animal blood transfusion; as well as in perfusion for artificial organs.
In order to serve the above indicated functions, a blood substitute must provide the essential capacity to carry and release oxygen in an animal body. Current research attention in the development of suitable blood substitute has been focusing on utilizing hemoglobin as the base material, by reprocessing the same using polymerization or encapsulation technique so that it can be used. There have also been research efforts in developing plasma substitutes prepared from artificially synthesized oxygen-carrying material. However, at the present time, the artificially prepared oxygen-carrying material has not been able to provide the same level of functionality as the naturally occurring hemoglobin, and thus is unable to satisfy the physiological oxygen transport requirement in animal bodies.
Hemoglobin existing in outdated human or animal bloods has been under continuous evaluation for its potential application as a blood substitute. Separation and purification of hemoglobin from mammalian bloods and reprocessing them into oxygen carrying red blood cells provide an immense potential in supplying large quantities of blood substitute. Regardless of its source, the hemoglobin must go through a series of separation steps to remove protein contaminants and stroma (containing phospholipids), which, among other things, can stimulate human body and cause undesirable side effects, and thus increase the purity of hemoglobin.
A number of methods for hemoglobin purification have been available in the course of the development of blood substitute. In the early stages, high-speed centrifugation and ultrafiltration were the most widely used techniques. These methods, however, can only provide a very crude separation; many contaminants still exist in the hemoglobin solution after the purification treatment.
In 1967, Rabiner developed a method for hemoglobin purification, Rabiner, S. F., et al.: Evaluation of a stroma-Free hemoglobin solution for use as a plasma expander, J. Exp. Med. 126, 1127-1142 (1967). First, red blood cells were separated from outdated human whole blood by centrifugation and then washed with saline. Distilled water or organic solvents with low osmotic pressure were then added thereto to hemolyze the red blood cells. Hemoglobin would be released as a result of hemolysis of the red blood cells. Then the solution was subject to a high-speed centrifugation at 35,000 g to precipitate and remove cell membrane stroma. Finally the solution was filtered through 0.1 .mu.m filter membrane to obtain purified hemoglobin. The Rabiner method is only capable of removing stromal particles that are relatively large; it, however, does not remove other proteins in the red blood cell and smaller cell membrane particles.
Drabkin and DeVenuto respectively developed crystallization methods for hemoglobin purification, both of which involved the first step of separating plasma and washing blood cells see, DeVenuto, F., et al.: Characteristics of stroma-free hemoglobin prepared by crystallization, J. Lab. Clin. Med. 89: 509-16 (1977). Then the red blood cells were hemolyzed to release hemoglobin. A relatively low-speed centrifugation, at 4,000 g, was applied to the hemolysate to remove relatively larger stromal particulate matter. Then a high concentration phosphate buffer was added to the solution to salt out hemoglobin, which is then subject to a crystal growth to obtain high purity hemoglobin. Because the crystal growth is a very time-consuming process, mass production utilizing this technique is difficult, and it is not practical to use this process to obtain large amount of high purity hemoglobin.
Cheung in 1983 utilized the principle of chromatography to develop a technique for hemoglobin purification using DEAE cellulose. In an article entitled "The Preparation of Stroma-Free Hemoglobin by Selective DEAE-Cellulose Absorption," Analytical Biochemistry 137, 481-484 (1984), by Cheung et al., it is disclosed that DEAE-Cellulose was used to absorb negatively charged proteins. By controlling the solution pH at a basic value (pH 7.5), proteins with an isoelectric point (pI) of far less than 7.5 can be selectively absorbed on the cellulose. On comparison, hemoglobin, which has a pI of about 7 and is only slightly negatively charged, will not be absorbed by the cellulose. Therefore, after mixing and absorption reach equilibrium and a series of filtration steps to remove the stroma-containing cellulose, purified hemoglobin can be obtained. However, because cellulose resins are relatively expensive, this process is not economical in practical applications, unless the cellulose resins can be reused.
In an article entitled "A Continuous-Flow High-Yield Process for Preparation of Lipid-Free Hemoglobin," Analytical Biochemistry 137, 191-198 (1986), Deloach proposed a technique which combined dialysis and ultrafiltration to completely remove phospholipids. Phospholipids often cause strong immune reaction or side effects after blood transfusion. In this technique, the red blood cell was subject to controlled dialysis to gradually change the osmotic pressure thereof to prevent severe osmotic shock from red blood cells swelling. However, the dialytic procedure also made red blood cell membranes permeable to hemoglobin and would allow hemoglobin to be released from inside the red blood cell without rupturing the cell membrane. The dialyzed red blood cell solution was processed through a 0.1 .mu.m pore to remove red blood cell membrane (or the so-called RBC-ghost) and obtain a phospholipid-free hemoglobin solution. This technique, however, does not remove other protein contaminants in the red blood cells.
More recently, Christensen et al., in an article entitled "Preparation of Human Hemoglobin Ao for Possible Use as A Blood Substitute," J. Biochemical and Biophysical Methods 17, 143-154 (1988), developed chromatographic methods for hemoglobin purification using anion-exchanger cellulose, such as DEAE, or cation-exchanger cellulose, such as CM. During the chromatographic separation, the pH or ionic strength of the eluent can be adjusted to control the type of proteins that can be eluted. Although this technique involves relative advanced technology, it is quite expensive and will not be economically justified unless the products have very high added value.
In 1990, Pristoupil used chloroform to extract hemoglobin. See, Pristoupil, et al., "Stroma-Free Hemoglobin Solutions Purified by Chloroform and Pasteurization", International Journal of Artificial Organs, vol. 13, no. 8, 383-387 (1990). Red blood cells, after being separated from plasma, was added to a separation phase containing sodium phosphate buffer (pH 7.5) and chloroform. The red blood cells were hemolyzed to release hemoglobin, which was then dissolved into the aqueous phase containing the phosphate buffer. The hydrophobic stroma and other non-heme proteins would precipitate in the chloroform phase. After charcoal adsorption and filtration through a 0.22 .mu.m membrane, a purified hemoglobin can be obtained. The purification procedure can be achieved with a relatively high speed and simplicity. However, any residual chloroform can be toxic to the human body, and safety will always be a concern using the extraction technique.